Use of Parapox B2L protein to modify immune responses to administered antigens

ABSTRACT

The  Parapox  B2L virus envelope protein is used as an adjuvant to enhance a subject&#39;s response to an administered antigen. Both antibody and cellular immune responses can be modified. B2L protein is particularly useful as an adjuvant for poorly immunogenic tumor vaccines and subunit vaccines, such as those useful for preventing and/or treating flu, tuberculosis, respiratory syncytial virus, anthrax and HIV.

[0001] This application is a continuation under 35USC120 of U.S. Ser. No. 10/414,609, filed Apr. 15, 2003, which is a continuation of U.S. Ser. No. 10/123,058, filed on Apr. 15, 2002, and is a continuation in part of PCT Appl No. PCT/US02/38971, filed Dec. 6, 2002, which claims the benefit of U.S. provisional application Ser. No. 60/336,694 filed Dec. 7, 2001.

[0002] This invention was funded in part by DARPA Grant No N652369915426, Account number 36001, by which the U.S. Government may have certain rights in this invention.

FIELD OF THE INVENTION

[0003] The invention relates to the use of B2L and PP30 proteins of a Parapox virus as a vaccine adjuvant.

BACKGROUND OF THE INVENTION

[0004] Cutaneous Parapoxvirus ovis causes recruitment of epidermal dendritic cells to the infection site in sheep and subsequent cell-mediated immunity (Lear et al., Eur. J. Dermatol. 6, 135-40, 1996; Haig et al., Comp Immun. Microbiol. Infect. Dis. 20 197-204, 1997). Attenuated Parapox viruses can be used to induce Paradox-specific immunity. U.S. Pat. No. 6,162,600. In addition, the highly attenuated strain D1701 (Baypamun HK®) is used as a non-specific immunomodulator (Buttner et al., Immunol. Microbiol. Infect. Dis. 16, 1-10, 1993) to promote immunity to heterologous pathogens.

[0005] Attenuation of Parapox virus, however, is time-consuming, taking from 100 to 200 culture passages; according to WO 95/22978, it takes from three to five years to perform each 100 passages, depending on the species of virus used. Attenuation can, therefore, “encompass a period lasting from ten to twenty years.” See WO 95/22978, page 9.

[0006] WO 95/22978 discloses the use of combinations of two or more individual Parapox virus components as “multipotent paramunity inducers” for use as adjuvant therapy for tumors and the prevention of metastases. The components can be individual polypeptides or detached envelopes of poxviruses. WO 95/22978, however, does not disclose any particular viral polypeptides other than the viral fusion protein and adsorption protein. Moreover, WO 95/22978 teaches that the disclosed paramunity inducers have virtually no immunogenic properties.

[0007] There is a need in the art for simple, effective vaccine adjuvants that can be used to enhance immune responses against tumors and dysplastic lesions and against exogenous pathogens.

SUMMARY OF THE INVENTION

[0008] It is an object of the invention to provide reagents and methods for modifying immune responses to administered antigens. This and other objects of the invention are provided by one or more of the embodiments described below.

[0009] One embodiment of the invention provides a method of enhancing an immune response to a vaccine composition. The method involves administering to a subject in need thereof (a) an effective amount of a B2L viral envelope protein of a Parapox virus and (b) a vaccine composition comprising an active component. The adjuvant B2L viral envelope protein thereby enhances the immune response to the vaccine composition.

[0010] Another embodiment of the invention provides a pharmaceutical composition comprising a B2L viral envelope protein of a Parapox virus and a vaccine composition comprising an active component.

[0011] Yet another embodiment of the invention provides a pharmaceutical composition comprising a nucleic acid molecule encoding a B2L viral envelope protein of a Parapox virus and a vaccine composition comprising an active component.

[0012] Thus, the invention provides pharmaceutical compositions and methods using B2L protein to modify immune responses to administered antigens.

DETAILED DESCRIPTION OF THE INVENTION

[0013] The invention is based on the ability of a Parapox viral envelope protein termed “B2L” to act as an adjuvant, i.e., to augment or otherwise modify a subject's immune response to an administered antigen and/or an active component of a vaccine. As noted below, the descriptions herein are also applicable to a second Parapox immunostimulatory polypeptide, PP30, and references herein to B2L are understood to encompass both proteins, absent contrary implication. Administered antigens include, but are not limited to, cells expressing tumor antigens, attentuated or killed pathogens and antigenic components thereof or nucleic acids encoding the antigenic components.

[0014] Both antibody and cellular immune responses can be modified. B2L protein is particularly useful as an adjuvant for poorly immunogenic tumor antigens and subunit vaccines, such as those useful for preventing and/or treating flu, tuberculosis, respiratory syncytial virus, anthrax and HIV.

[0015] B2L is the second open reading frame in the BamHl B fragment of the Orf virus genome (Sullivan et al., Virology 202, 968-73, 1994). Prior work teaches that, as the activity of epitopes responsible for antigen-specific immunization decrease, adjuvant activity of the preparations increases. See WO 95/22978, page 4. B2L is an immunogenic protein; in fact, B2L protein is one of a few Orf virus proteins to which a strong antibody response can be mounted in sheep. Sullivan et al., 1994. Thus, it is surprising that purified B2L protein itself has adjuvant activity.

[0016] B2L proteins for use in the compositions and methods described herein are those of the Parapoxvirus genus, such as Orf virus (OV), particularly the Parapox ovis strains NZ2, NZ7, NZ10, and D1701. Orf viruses are reviewed in Robinson & Balassu, Vet. Bull 51, 771, 1981; Robinson & Lyttle, in Binns & Smith, eds., recombinant poxviruses, Chapter 9, pp. 306-17, CRC Press, Boca Raton, 1992. An amino acid sequence for the B2L protein of OV NZ2 is disclosed in Sullivan et al., Identification and characterization of an orf virus homologue of the vaccinia virus gene encoding the major envelope antigen p37K, Virology 202 (2), 968-73, 1994, and is shown in SEQ ID NO:2. A coding sequence for SEQ ID NO:2 is shown in SEQ ID NO:1. The amino acid sequences of the B2L proteins obtained from D1701 and NZ2 are highly conserved. The amino acid sequence of the D1701 protein is shown in SEQ ID NO:4. A coding sequence for SEQ ID NO:4 is shown in SEQ ID NO:3.

[0017] Purified B2L protein is separated from other compounds that normally associate with the B2L protein in the virus, such as other envelope components. A preparation of purified B2L protein is at least 80% pure; preferably, the preparations are 90%, 95%, or 99% pure. Purity of the preparations can be assessed by any means known in the art, such as SDS-polyacrylamide gel electrophoresis.

[0018] Purified B2L protein for use in compositions and methods of the invention can be purified from Parapox viruses or from cells infected by the viruses, by recombinant DNA methods, and by chemical synthesis. Purification methods include, but are not limited to, size exclusion chromatography, ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography, and preparative gel electrophoresis.

[0019] B2L protein can be expressed recombinantly, after insertion of B2L coding sequences into an expression vector that contains the necessary elements for the transcription and translation of the inserted coding sequence. Maintenance of orf viruses in culture is disclosed in WO 97/37031. A preferred system for maintaining and expressing B2L protein is HKB11 cells transfected with B2L in a vector such as a p2ToP, pCEP4, or pcDNA3.1 vector (Invitrogen). Recombinantly produced B2L protein can be secreted into the culture medium and purified. Methods for producing proteins recombinantly are well known to those skilled in the art.

[0020] A B2L protein also can be produced using chemical methods to synthesize its amino acid sequence, such as by direct peptide synthesis using solid-phase techniques (Merrifield, J. Am. Chem. Soc. 85, 2149-2154, 1963; Roberge et al., Science 269, 202-204, 1995). Protein synthesis can be performed using manual techniques or by automation. Optionally, fragments of a B2L protein can be separately synthesized and combined using chemical methods to produce a full-length molecule.

[0021] “B2L protein” as used herein includes both functional portions of B2L and full-length or partial biologically active B2L variants. Biologically active variants (i.e., variants that possess adjuvant activity) comprise amino acid substitutions, insertions, and/or deletions with respect to the amino acid sequences shown in SEQ ID NOS:2 or 4. Amino acid substitutions are defined as one for one amino acid replacements. They are conservative in nature when the substituted amino acid has similar structural and/or chemical properties. Examples of conservative replacements are substitution of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine. Biologically active variants can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, 20, 25, or 30 or more conservative amino acid substitutions as long as adjuvant activity of the B2L variant is maintained.

[0022] Amino acid insertions or deletions are changes to or within an amino acid sequence. They typically fall in the range of about 1 to 5 amino acids (i.e., 1, 2, 3, 4, or 5). Guidance in determining which amino acid residues can be substituted, inserted, or deleted without abolishing biological or immunological activity of a B2L protein can be found using computer programs well known in the art, such as DNASTAR software. Biological activity of a B2L protein having an amino acid substitution, insertion, and/or deletion can be tested, for example, as described in Example 1.

[0023] Functional portions of B2L comprising, for example, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 360, 370, 375, or 377 amino acids, also can be used in the compositions and methods of the invention, provided that the portions of B2L retain biological activity, e.g., the ability to enhance an immune response and/or exert a chemotactic effect on enriched dendritic cell populations.

[0024] Purified B2L protein can be used in pharmaceutical compositions. Pharmaceutical compositions of the invention can be used to boost immune responses in mammals, including laboratory animals (e.g., mice, rats, hamsters, guinea pigs), companion animals (e.g., dogs, cats), farm animals (e.g., horses, cows, sheep, pigs, goats), and humans.

[0025] Pharmaceutical compositions of the invention include a pharmaceutically acceptable carrier. Typically these will be sterile formulations in a diluent or vehicle that is free of pyrogenic components. Buffers, stabilizers, and the like can be included, as is known in the art. Optionally, pharmaceutical compositions include conventional adjuvants, such as aluminum hydroxide and aluminum phosphate (collectively commonly referred to as alum), saponins complexed to membrane protein antigens (immune stimulating complexes), pluronic polymers with mineral oil, killed mycobacteria in mineral oil, Freund's complete adjuvant, bacterial products, such as muramyl dipeptide, and lipopolysaccharides.

[0026] If desired, B2L protein can be coupled to an antigen. Means of making such molecules are well known in the art. For example, coupled molecules can be synthesized chemically or produced by covalently linking two polypeptide segments or by standard procedures in the art of molecular biology. Recombinant DNA methods can be used to prepare fusion proteins, for example, by making a DNA construct which comprises B2L coding sequences in proper reading frame with nucleotides encoding a polypeptide to be coupled with B2L and expressing the DNA construct in a host cell, as is known in the art. Many kits for constructing fusion proteins are available from companies such as Promega Corporation (Madison, Wis.), Stratagene (La Jolla, Calif.), CLONTECH (Mountain View, Calif.), Santa Cruz Biotechnology (Santa Cruz, Calif.), MBL International Corporation (MIC; Watertown, Mass.), and Quantum Biotechnologies (Montreal, Canada; 1-888-DNA-KITS).

[0027] B2L-containing compositions are co-administered with a particular antigen or vaccine composition. “Co-administration” includes administration of B2L and the antigen or vaccine composition separately or in the same composition.

[0028] Vaccine compositions comprise one or more active components, e.g., a tumor antigen or an attenuated or killed pathogen or an antigenic component thereof. Antigenic components include any component that is recognized by cells of the immune system. Suitable tumor antigens include, but are not limited to, α-fetoprotein, BAGE, β-HCG, CEA, ESO,, GAGE, gangliosides, Her-2/neu, HPV E6/E7, immunoglobulins, MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-12, MART-1, Melan-A, melanoma antigen gp75, gp100, MN/G250, MUC1, MUC2, MUC3, MUC4, MUC18, PSA, PSM, RAGE, ras, SART-1, telomerase, thyroperoxidases, tyrosinases, and p53.

[0029] Vaccine compositions also can include a pathogen. The pathogen can be, e.g., an attenuated or killed virus, bacterium, mycoplasm, parasite, yeast, fungus, prion, or a protozoan. Suitable pathogens include human immunodeficiency viruses, Herpes viruses, hepatitis viruses, pox viruses, flu viruses, measles, mumps, rubella, rabies, respiratory syncytial viruses, Bacillus anthracis, Bordetella pertussis, Borrelia burgdorferi, Clostridium tetani, Corynebacterium diphtheriae, Haemophilus influenza B, Neisseria meningitidis, Salmonella typhi, Streptococcus pneumoniae, and Vibrio cholerae. The active component also can be, for example, an immunogenic fragment, extract, subunit, metabolite, or recombinant construct of such a pathogen. Optionally, the active component can be mixed with a pharmaceutically acceptable carrier and/or a conventional adjuvant, as described above.

[0030] Adjuvant compositions comprising B2L protein can be administered sequentially or simultaneously with a vaccine composition, including a poorly immunogenic subunit vaccine. If desired, the B2L protein can be present in the vaccine composition. Suitable routes of administration include, without limitation, subcutaneous, intravenous, nasal, ophthalmic, transdermal, intramuscular, intradermal, intragastric, perlingual, alveolar, gingival, intraperitoneal, intravaginal, pulmonary, rectal, and oral administration. Administration can be by any suitable means, including injection, topical administration, ingestion, or inhalation. Single and/or multiple administrations are contemplated.

[0031] Optionally, B2L protein can be administered using a nucleic acid molecule encoding the protein. The nucleic acid molecule can be either DNA, RNA, or a DNA/RNA chimera. Use of DNA-encoded elicitors of immune responses is discussed, for example, in McDonnel & Askari, New Engl J. Med. 334, 42-45, 1996; Robinson, Can. Med. Assoc. J. 152, 1629-32, 1995; Fynan et al., Int. J. Immunopharmacol. 17, 79-83, 1995; Pardoll & Beckerleg, Immunity 3, 165-69, 1995, and Spooner et al., Gene Therapy 2, 173-80, 1995. Chimeric RNA/DNA oligonucleotide based gene therapy is discussed in Lai & Lien, Expert Opin Biol Ther 2001 January; 1(1):41-7.

[0032] A variety of delivery systems, both viral and non-viral, can be used to administer the B2L-encoding nucleic acid molecule. Such delivery systems include, but are not limited to, naked plasmid DNA, viral expression vectors, and nucleic acid molecules in conjunction with a liposome or a condensing agent. See U.S. Pat. No. 6,303,372.

[0033] The determination of a therapeutically effective dose of B2L is well within the capability of those skilled in the art. A therapeutically effective dose refers to that amount of active ingredient (i.e., B2L protein or nucleic acid encoding B2L) that results in an augmentation of the immune response to a co-administered antigen, compared to that which occurs in the absence of the therapeutically effective dose.

[0034] The therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rats, rabbits, dogs, or pigs. The animal model also can be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.

[0035] Therapeutic efficacy and toxicity, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population), can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50.

[0036] Pharmaceutical compositions that exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies are used in formulating a range of dosage for human use. The dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.

[0037] Suitable dosages and treatment regimens for administration of either B2L protein or nucleic acid molecules encoding B2L include, but are not limited to, daily, twice-or three-times weekly, weekly, bi-weekly, monthly, bimonthly, or yearly treatments of about 1, 5, 10, 15, 20, 25, 50, 75, 100, or 200 ug/m² (or approximately 0.05, 0.1, 0.2, 0.25, 0.3, 0.5, 0.75, 1, 2, 5, or 10 mg/kg). Preferred routes of administration include subcutaneous, intradermal, intramuscular administration. The exact dosage, treatment regimen, and route of administration, however, will be determined by the practitioner, in light of factors related to the subject that requires treatment.

[0038] All patents, patent applications, and references cited in this disclosure are expressly incorporated herein by reference in their entireties. The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific example, which is provided for purposes of illustration only and is not intended to limit the scope of the invention.

EXAMPLES

[0039] B2L is an immunostimulatory Parapox protein. Using PCR amplification of segments of the parapox genome, (D1701 strain), we identified full-length coding sequences from open reading frames (ORFs) of Parapox. Specific primers were synthesized to the ORFs generating individual PCR products. Utilizing linear expression element (LEE) technology, the PCR products were tested for in vivo DC recruiting activity. DNA was precipitated on gold beads and the complexes were introduced by biolistic inoculation into mouse abdominal skin that had been prepared by treatment with shears and Nair depilatory. Each mouse was treated with 4-6 shots of the DNA-gold complexes. The recruitment of dendritic cells to the site of inoculation was assessed after 4 days. To assess the dendritic cell recruitment, the mice were euthanized, the location of the inoculations marked and the abdominal skin was harvested. Inoculation sites were isolated and trimmed of surrounding tissue. Skin segments were immersed in cryoprotective medium and quickly frozen in liquid nitrogen. Thin-sections were cut and stored at −20° C. for subsequent fixation and staining. The presence of dendritic cells (FITC-labeled, anti-I-A^(d)/I-E^(d) antibody positive cells with dendritic morphology) was evaluated by fluorescence microscopy. We identified B2L by its dendritic cell recruitment activity in this assay, with further observations of the best humoral immune response occurring at the inoculation site.

[0040] B2L is an adjuvant for generating a humoral immune response. Mice were inoculated with a low dose (0.5 μgDNA/ear; 1 μg total/mouse) of a plasmid encoding human ∞₁-anti-trypsin (hAAT). When inoculated by itself, the low dose is below the threshold of antibody induction with a single immunization. However, when administered in the presence of a genetic adjuvant, such as a plasmid encoding GM-CSF, an antibody response to HAAT is observed in most mice. The addition of LEEs encoding B2L enhanced the generation of an antibody response to hAAT. In fact, the animals that received the B2L LEEs along with the HAAT antigen exhibited an earlier antibody response than the mice that received GM-CSF as an adjuvant. The responses of the 2 groups were similar with respect to the levels of circulating antibodies to HAAT that were obtained without a booster inoculation.

[0041] B2L enhances antibody responses to multiple antigens. In these experiments, B2L was co-inoculated with plasmids encoding hAAT and hepatitis B surface antigen (HbsAg). The level of each antigen-encoding plasmid was reduced to 0.2 μg/inoculation with B2L or control plasmid at 1 μg/inoculation. B2L enhanced the generation of antibodies in response to both hAAT and HbsAg.

[0042] B2L adjuvants provide a protective immune response for influenza virus. Mice were immunized twice with plasmid encoding influenza hemagglutinin (pCMVi-HA). Mice were immunized with 4 levels (0.4-400 ng) of pCMVi-HA plasmid along with 1 μg control plasmid, pCMVi-mGM-CSF, or pCMVi-B2L. When challenged with influenza virus, mice that were immunized without adjuvant still succumbed to the infection. 50% of the mice that received the highest dose of antigen (400 ng/immunization) without adjuvant died upon challenge. In contrast, mice that were immunized with this antigen level in the presence of B2L were fully protected. This level of protection was also observed with the control adjuvant, mGM-CSF. In this initial experiment, B2L also enhanced the level of protection for mice that were immunized with only 40 ng pCMVi-HA. At this level, B2L exhibited a protective benefit compared to mGM-CSF.

[0043] PP30 is another Parapox immunostimulatory polypeptide. Screening the entire dendritic cell recruitment activity and adjuvant activity, we found a second immunostimulatory Parapox gene product, designated PP30. SEQ ID NO:6 is a PP30 amino acid sequence, and SEQ ID NO:5 is a native coding sequence of PP30 derived from Parapox strain D1701. PP30 is also found in alternative Parapox strains. In general, PP30 may be substituted for B2L in the compositions and methods disclosed herein, and the description and examples herein reciting B2L are applicable to PP30 as well, however, the adjuvant activity of PP30 is distinguishable from B2L as it stimulated an antibody response to hAAT significantly greater than that to HbsAg. PP30 is a newly identified gene with no apparent homolog in the NCBI database.

[0044] B2L and PP30 Parapox proteins are immunostimulatory across Parapox strains. By comparing the sequences of B2L from the D1701 strain to the related gene in the NZ2 strain we identified sequence differences at both the nucleotide and predicted protein levels. Therefore, we isolated DNA from the NZ2 strain of Parapox, amplified the NZ2-B2L gene by PCR and cloned this into pCMVi plasmid. The efficacy of NZ2-B2L and D1701-B2L was then compared and showed comparable dendritic cell recruitment activity and adjuvant activity. Cross-strain immunostimulatory activity was similarly shown for PP30.

[0045] Direct B2L protein administration enhances specific antibody response. To demonstrate the effect of the B2L protein on the generation of a specific antibody response against HBsAg, protein mixtures without alum were injected into groups of mice:

[0046] Group 1: 1 μg HBsAg protein in PBS (“antigen only” control)

[0047] Group 2: 1 μg HBsAg protein+3 μl Baypamum® in PBS (comparison with whole killed Parapox virus)

[0048] Group 3: 1 μg HBsAg protein+50 ng B2L-H6 in PBS (testing B2L protein as an adjuvant; note that the protein was expressed as a 6His fusion protein).

[0049] Group 4: 1 μg HBsAg protein+50 ng SRB4-H6 (comparison with an irrelevant negative control protein that is also a 6His fusion protein).

[0050] Group 5: 1 μg HBsAg protein+2.5 μl alum in PBS, mixed together and left on ice for 30 minutes beforehand (comparison with alum, the only FDA-approved adjuvant).

[0051] Each mouse was injected with 25 μl into the tibialis anterior muscle, and blood was collected before injection, and at each of the time points indicated. Sera were processed and stored at −20° C. Anti-HBsAg antibody levels were measured by ELISA. Groups 4 and 5 showed a modest (approximately 2-fold) increase in antibody elicitation over groups 1 and 2 at multiple time-points from 30 to 60 days, whereas Group 3 (B2L) showed a much more dramatic (over a 5-fold) increase over the same controls.

[0052] Direct B2L protein administration stimulates dendritic cell recruitment. To demonstrate B2L protein promotion of dendritic cell recruitment, aliquots containing 50 ng of B2L protein were injected into the skin of a mouse. A control mouse was injected with PBS alone in a comparable volume. The injected skin was harvested 4 days later. Sections were stained for DC using the anti-MHC II antibody 2G9, revealing a dramatic B2L protein-enhanced increase in dendritic cell recruitment.

[0053] B2L has adjuvant activity when coadministered with a tumor antigen. Tumor peptides frequently are weak immunogens, and immune responses to them typically require that they be administered together with an adjuvant. A tumor vaccine consisting of an emulsion prepared with Montanide ISA 51 containing 100 ug of a melanoma peptide (TRP-2) and 100 ug of either V5His6-tagged B2L or and irrelevant protein (hSLC; human secondary lymphoid chemokine) was administered subcutaneously at the base of the tail three days after implantation of a melanoma tumor known to express the antigen included in the vaccine. Tumor growth was monitored until the nodules reached the volume permitted by an approved animal care and use protocol. By comparison with animals given the tumor antigen together with an irrelevant protein, there was a trend toward reduced tumor growth in mice treated with tumor antigen together with tagged B2L. The results are consistent with B2L's augmentation of an immune response to the TRP-2 tumor antigen, which slowed the growth of the tumor expressing this antigen.

1 6 1 1137 DNA PARAPOX 1 atgtggccgt tctcctccat ccccctgggc gccgactgcc gcgtcgtgga gacgctgccc 60 gcagaggtgg cgtctttggc gcagggcaac atgagcaccc tcgactgctt caccgctatc 120 gccgagtccg cgaagaagtt cttgtacatc tgcagcttct gctgcaacct gagctccacc 180 aaggagggcg tcgacgtcaa ggacaagctc tgcacgctcg ccaaggaggg cgtagacgtc 240 acgctgctcg tggacgtgca gagcaaggac aaggacgcgg acgagctgcg cgaggcgggc 300 gtcaactact acaaggtcaa ggtgtccacc aaggagggcg tcggcaacct tctcggcagc 360 ttctggctct cggacgccgg gcactggtac gtgggaagcg cctcgctcac gggcgggtcc 420 gtgtccacca tcaagaacct cgggctctac tccaccaaca agcacctggc ctgggacctc 480 atgaaccgct acaacacctt ctactccatg atcgtggagc cgaaggtgcc gttcacgcgg 540 ctctgctgcg ccatcgtcac gcccacggcc acgaacttcc acctcgacca ctccgggggc 600 ggcgtattct tctcggactc gccggagcgc ttcctaggct tctaccgcac gctcgacgag 660 gacctcgtgc tgcaccgcat cgagaacgcc aagaacagca tcgacctctc gctgctctcg 720 atggtgccgg tgatcaagca cgccagcgcc gtggagtact ggccgcagat cattgacgcg 780 ctgctgcgcg cggccatcaa ccgcggcgtg cgcgtgcgcg tgatcattac cgagtggaag 840 aacgcggacc cgctttcggt ctcggccgcg cgcagcctcg acgactttgg cgtcggcagc 900 gtggacatgt ccgtgcgcaa gttcgtggta cccggccggg acgacgccgc gaacaacact 960 aagctgctca tcgtggacga caccttcgcg cacctcacgg tcgccaacct cgacggcacg 1020 cactaccgct accacgcctt cgtgagcgtg aacgccgaga agggcgacat cgtcaaggac 1080 ctgtccgcgg tcttcgagcg ggactggcgc tcggagttct gcaagccaat aaattaa 1137 2 378 PRT PARAPOX 2 Met Trp Pro Phe Ser Ser Ile Pro Leu Gly Ala Asp Cys Arg Val Val 1 5 10 15 Glu Thr Leu Pro Ala Glu Val Ala Ser Leu Ala Gln Gly Asn Met Ser 20 25 30 Thr Leu Asp Cys Phe Thr Ala Ile Ala Glu Ser Ala Lys Lys Phe Leu 35 40 45 Tyr Ile Cys Ser Phe Cys Cys Asn Leu Ser Ser Thr Lys Glu Gly Val 50 55 60 Asp Val Lys Asp Lys Leu Cys Thr Leu Ala Lys Glu Gly Val Asp Val 65 70 75 80 Thr Leu Leu Val Asp Val Gln Ser Lys Asp Lys Asp Ala Asp Glu Leu 85 90 95 Arg Glu Ala Gly Val Asn Tyr Tyr Lys Val Lys Val Ser Thr Lys Glu 100 105 110 Gly Val Gly Asn Leu Leu Gly Ser Phe Trp Leu Ser Asp Ala Gly His 115 120 125 Trp Tyr Val Gly Ser Ala Ser Leu Thr Gly Gly Ser Val Ser Thr Ile 130 135 140 Lys Asn Leu Gly Leu Tyr Ser Thr Asn Lys His Leu Ala Trp Asp Leu 145 150 155 160 Met Asn Arg Tyr Asn Thr Phe Tyr Ser Met Ile Val Glu Pro Lys Val 165 170 175 Pro Phe Thr Arg Leu Cys Cys Ala Ile Val Thr Pro Thr Ala Thr Asn 180 185 190 Phe His Leu Asp His Ser Gly Gly Gly Val Phe Phe Ser Asp Ser Pro 195 200 205 Glu Arg Phe Leu Gly Phe Tyr Arg Thr Leu Asp Glu Asp Leu Val Leu 210 215 220 His Arg Ile Glu Asn Ala Lys Asn Ser Ile Asp Leu Ser Leu Leu Ser 225 230 235 240 Met Val Pro Val Ile Lys His Ala Ser Ala Val Glu Tyr Trp Pro Gln 245 250 255 Ile Ile Asp Ala Leu Leu Arg Ala Ala Ile Asn Arg Gly Val Arg Val 260 265 270 Arg Val Ile Ile Thr Glu Trp Lys Asn Ala Asp Pro Leu Ser Val Ser 275 280 285 Ala Ala Arg Ser Leu Asp Asp Phe Gly Val Gly Ser Val Asp Met Ser 290 295 300 Val Arg Lys Phe Val Val Pro Gly Arg Asp Asp Ala Ala Asn Asn Thr 305 310 315 320 Lys Leu Leu Ile Val Asp Asp Thr Phe Ala His Leu Thr Val Ala Asn 325 330 335 Leu Asp Gly Thr His Tyr Arg Tyr His Ala Phe Val Ser Val Asn Ala 340 345 350 Glu Lys Gly Asp Ile Val Lys Asp Leu Ser Ala Val Phe Glu Arg Asp 355 360 365 Trp Arg Ser Glu Phe Cys Lys Pro Ile Asn 370 375 3 1137 DNA PARAPOX 3 atgtggccgt tctcctccat ccccgtgggc gcccaatgcc gcgtcttgga aacgctgccc 60 gcagaggtgg cgtccctggc gcagggcaac atgagcaccc tcgactgctt caccgccatc 120 gccgagtccg cgaagaaatt tttgtacatc tgcagcttct gctgcaacct gagctccacc 180 aaggagggcg tcgacgtcaa agacaagctc tgcacgctcg ccaaggaagg cgttgacgtc 240 acgctgctcg tggacgtgca gagcaaggac aaggacgcgg acgaactgcg cgcggcgggc 300 gtcaactact acaaggtcaa agtgtccacg cgggaaggcg tcggcaacct tctcggcagc 360 ttctggctct cggacgccgg gcactggtac gtgggcagcg cctcgctcac gggcgggtcc 420 gtgtccacca tcaagaacct cgggctctac tccaccaaca agcacctggc ctgggacctc 480 atgaaccgct acaacacctt ctactccatg atcgtggagc cgaaggtgcc gttcacgcgg 540 ctctgctgcg ccgtcgtcac gcccacggcc acgaacttcc acctcaacca ctccgggggc 600 ggcgtattct tctcggactc gccggagcgc ttcctaggct tctaccgcac gctcgacgag 660 gacctcgtgc tgcaccgcat cgagaacgcc aagaacagca tcgacctctc gctgctctcg 720 atggtgccgg tgatcaagca cgccggcgcc gtggagtact ggccgcggat catagacgcg 780 ctgctgcgcg cggccatcaa ccgcggcgtg cgcgtgcgcg tgatcatcac cgagtggaag 840 aacgcggacc cgctgtcggt ctcggccgcg cgcagcctcg acgactttgg cgtcggtagc 900 gtggacatgt ccgtgcgcaa gttcgtggta cccggccggg acgacgctgc gaacaacacc 960 aagctgctta tcgtggacga caccttcgcg cacctcacgg tcgccaacct cgacggcacg 1020 cactaccgct accacgcctt cgtgagcgtg aacgccgaga agggcgacat cgtcaaggac 1080 ctgtccgcgg tcttcgagcg ggactggcgc tcggagtttt gcaagccaat aaattaa 1137 4 378 PRT PARAPOX 4 Met Trp Pro Phe Ser Ser Ile Pro Leu Gly Ala Gln Cys Arg Val Leu 1 5 10 15 Glu Thr Leu Pro Ala Glu Val Ala Ser Leu Ala Gln Gly Asn Met Ser 20 25 30 Thr Leu Asp Cys Phe Thr Ala Ile Ala Glu Ser Ala Lys Lys Phe Leu 35 40 45 Tyr Ile Cys Ser Phe Cys Cys Asn Leu Ser Ser Thr Lys Glu Gly Val 50 55 60 Asp Val Lys Asp Lys Leu Cys Thr Leu Ala Lys Glu Gly Val Asp Val 65 70 75 80 Thr Leu Leu Val Asp Val Gln Ser Lys Asp Lys Asp Ala Asp Glu Leu 85 90 95 Arg Ala Ala Gly Val Asn Tyr Tyr Lys Val Lys Val Ser Thr Arg Glu 100 105 110 Gly Val Gly Asn Leu Leu Gly Ser Phe Trp Leu Ser Asp Ala Gly His 115 120 125 Trp Tyr Val Gly Ser Ala Ser Leu Thr Gly Gly Ser Val Ser Thr Ile 130 135 140 Lys Asn Leu Gly Leu Tyr Ser Thr Asn Lys His Leu Ala Trp Asp Leu 145 150 155 160 Met Asn Arg Tyr Asn Thr Phe Tyr Ser Met Ile Val Glu Pro Lys Val 165 170 175 Pro Phe Thr Arg Leu Cys Cys Ala Val Val Thr Pro Thr Ala Thr Asn 180 185 190 Phe His Leu Asn His Ser Gly Gly Gly Val Phe Phe Ser Asp Ser Pro 195 200 205 Glu Arg Phe Leu Gly Phe Tyr Arg Thr Leu Asp Glu Asp Leu Val Leu 210 215 220 His Arg Ile Glu Asn Ala Lys Asn Ser Ile Asp Leu Ser Leu Leu Ser 225 230 235 240 Met Val Pro Val Ile Lys His Ala Gly Ala Val Glu Tyr Trp Pro Arg 245 250 255 Ile Ile Asp Ala Leu Leu Arg Ala Ala Ile Asn Arg Gly Val Arg Val 260 265 270 Arg Val Ile Ile Thr Glu Trp Lys Asn Ala Asp Pro Leu Ser Val Ser 275 280 285 Ala Ala Arg Ser Leu Asp Asp Phe Gly Val Gly Ser Val Asp Met Ser 290 295 300 Val Arg Lys Phe Val Val Pro Gly Arg Asp Asp Ala Ala Asn Asn Thr 305 310 315 320 Lys Leu Leu Ile Val Asp Asp Thr Phe Ala His Leu Thr Val Ala Asn 325 330 335 Leu Asp Gly Thr His Tyr Arg Tyr His Ala Phe Val Ser Val Asn Ala 340 345 350 Glu Lys Gly Asp Ile Val Lys Asp Leu Ser Ala Val Phe Glu Arg Asp 355 360 365 Trp Arg Ser Glu Phe Cys Lys Pro Ile Asn 370 375 5 408 DNA PARAPOX 5 atggacgcgg tgtccgcgct ctgcgtgagc cctcgcggca gccgccgcca tgttcgtggc 60 gctgcagctc tgggccgtct acgagaacta cgacaacatc cgcgagttca acgcggcgaa 120 cgcggcgctg gagttcgcgc gcacggcggg cggcccgcgc gtggaccggc gcgtgttcga 180 ccccaacgac gaggccttcg acgtgcgcaa gaagtggcgc tgcgtgctct tcaagggcgc 240 ggcggtggcg gcctcggagt tcgggtttcg ttcgcacgac ggcgtctcgc ccgcgcgctt 300 cgcgagcgtg ggaggcgtgt gcggacgaga tcttcacggc cgcgagcgac ttccgcgtgt 360 tcaacccgtg tttgccgcca aatcagggca gcgaggcctg cctttttc 408 6 136 PRT PARAPOX 6 Met Asp Ala Val Ser Ala Leu Cys Val Ser Pro Arg Gly Ser Arg Arg 1 5 10 15 His Val Arg Gly Ala Ala Ala Leu Gly Arg Leu Arg Glu Leu Arg Gln 20 25 30 His Pro Arg Val Gln Arg Gly Glu Arg Gly Ala Gly Val Arg Ala His 35 40 45 Gly Gly Arg Pro Ala Arg Gly Pro Ala Arg Val Arg Pro Gln Arg Arg 50 55 60 Gly Leu Arg Arg Ala Gln Glu Val Ala Leu Arg Ala Leu Gln Gly Arg 65 70 75 80 Gly Gly Gly Gly Leu Gly Val Arg Val Ser Phe Ala Arg Arg Arg Leu 85 90 95 Ala Arg Ala Leu Arg Glu Arg Gly Arg Arg Val Arg Thr Arg Ser Ser 100 105 110 Arg Pro Arg Ala Thr Ser Ala Cys Ser Thr Arg Val Cys Arg Gln Ile 115 120 125 Arg Ala Ala Arg Pro Ala Phe Phe 130 135 

1. A method of enhancing an immune response to an antigen, comprising the steps of: administering to a subject mammal in need thereof (a) an effective amount of a B2L viral envelope protein of a Parapox virus, wherein the B2L protein is unassociated with other envelope components naturally associated with the B2L protein in the virus, and (b) an antigen, whereby the B2L protein acts as an adjuvant to enhance the immune response to the antigen; and detecting a resultant enhanced, specific immune response to the antigen.
 2. The method of claim 1 wherein the B2L protein and the antigen are administered sequentially.
 3. The method of claim 1 wherein the B2L protein and the antigen are administered simultaneously.
 4. The method of claim 1 wherein the B2L protein and the antigen are administered simultaneously as a fusion protein comprising the B2L protein and the antigen.
 5. The method of claim 1 wherein the B2L protein is administered by means of a nucleic acid encoding the B2L protein.
 6. The method of claim 1 wherein the antigen is administered by means of a nucleic acid encoding the antigen.
 7. The method of claim 1 wherein the antigen is administered as an attenuated or killed pathogen comprising the antigen.
 8. The method of claim 1 wherein the antigen is a tumor antigen.
 9. The method of claim 1 wherein the B2L protein and the antigen are administered by injection.
 10. The method of claim 1 wherein the B2L protein and the antigen are administered intradermally.
 11. The method of claim 1 wherein the Parapox virus is a Parapox virus ovis strain selected from the group consisting of NZ2, NZ7, NZ10, and D1701.
 12. The method of claim 1 wherein the mammal is a human.
 13. A pharmaceutical composition for enhancing an immune response to an antigen, the composition comprising: a B2L viral envelope protein of a Parapox virus, or a nucleic acid encoding said B2L protein, wherein the B2L protein is unassociated with other envelope components naturally associated with the B2L protein in the virus; and an antigen or a nucleic acid encoding said antigen, wherein administered to a subject mammal in need thereof, the B2L protein of the pharmaceutical composition acts as an adjuvant to enhance a specific immune response to the antigen.
 14. A pharmaceutical composition for enhancing an immune response to an antigen, the composition comprising: a PP30 protein of a Parapox virus, or a nucleic acid encoding said PP30 protein, wherein the PP30 protein is unassociated with other components naturally associated with the PP30 protein in the virus; and an antigen or a nucleic acid encoding said antigen, wherein administered to a subject mammal in need thereof, the PP30 protein of the pharmaceutical composition acts as an adjuvant to enhance a specific immune response to the antigen.
 15. A method of using a composition according to claim 14 to enhance an immune response to an antigen, comprising the steps of: administering to a subject mammal in need thereof (a) an effective amount of a PP30 protein of a Parapox virus, wherein the PP30 protein as found in Parapox strain D1701 comprises the amino acid sequence of SEQ ID NO:6, and wherein the PP30 protein is unassociated with other components naturally associated with the PP30 protein in the virus, and (b) an antigen, whereby the PP30 protein acts as an adjuvant to enhance the immune response to the antigen; and detecting a resultant enhanced, specific immune response to the antigen. 